PROJECT SUMMARY In the U.S. alone, up to 26 million people have chronic kidney disease, over 460,000 people are on dialysis, and 100,000 people await kidney transplants with 3,000 new patients added monthly. Given the growing lack of transplantable organs, patients typically require renal replacement therapies that themselves lead to substantial morbidity and mortality. We posit that biomanufactured kidney tissues, and ultimately, organs may offer an important solution to this growing problem. Indeed, recent protocols in developmental biology are unlocking the potential for stem cells to undergo differentiation and self-assembly to form ?mini-organs?, known as organoids. Kidney organoids exhibit remarkable tissue microarchitectures with high cellular density and heterogeneity akin to their in vivo counterparts. To bridge the gap from renal tissue building blocks to therapeutic organs, integrative approaches that combine bottom-up organoid assembly with top-down bioprinting are needed. It is difficult, if not impossible, to imagine how either organoids or bioprinting alone would fully replicate the complex multiscale features required for kidney function. Yet their combination could provide an enabling foundation for de novo organ manufacturing. To biomanufacture 3D vascularized kidney tissues for potential therapeutic applications, in Specific Aim 1, we will create microvascularized kidney organoids perfused by luminal connection with bioprinted macrovessels under controlled flow in vitro. We will produce iPSC-derived kidney organoids and subject them to fluid flow during their differentiation and maturation on an adherent extracellular matrix (ECM). We will then investigate the integration of their endogenous microvasculature with printed macrochannels by establishing a controlled VEGF gradient to guide anastomosis on a customized perfusion chip. This proof-of- concept experiment is a necessary first step towards a scalable tissue biomanufacturing process. In Specific Aim 2, we will evaluate the effects of macro-microvascular integration and perfusion on kidney organoid structure, glomerular filtration and tubular maturation. Both qualitative and quantitative analysis of vasculature and nephron development will be carried out to assess their morphology and function. We will then assess perfusion and transport through the vascular and tubular network(s) using a combination of bead flow, microCT, microperfusion, and micropuncture experiments. We will analyze the composition of primitive urine collected via a printed drainage channel co-localized near the organoid bed. To create 3D vascularized kidney tissues in a scalable manner, in Specific Aim 3, we will form tissue matrices composed of kidney organoids (1 mL or higher) within which a perfusable macrovasculature network will be patterned by embedded bioprinting. Kidney tissue viability, structure, maturation and function will be qualitatively and quantitatively analyzed following the protocols used in Specific Aim 2. If successful, our proposed discovery-based project will provide a foundational advance in kidney organ engineering for potential renal therapeutic applications.